Lipids in Health and Disease BioMed Central...Alzheimer's disease, and enhances cognitive function; and serve as endogenous anti-inflammatory molecules; and could be administered from

BioMed CentralLipids in Health and Disease

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Open AcceHypothesisEssential fatty acids and their metabolites could function as endogenous HMG-CoA reductase and ACE enzyme inhibitors, anti-arrhythmic, anti-hypertensive, anti-atherosclerotic, anti-inflammatory, cytoprotective, and cardioprotective moleculesUndurti N Das1,2
Address: 1UND Life Sciences, 13800 Fairhill Road, #321, Shaker Heights, OH 44120, USA and 2Department of Medicine, Bharati Vidyapeeth University Medical College, Pune, India

Email: Undurti N Das - [email protected]

AbstractLowering plasma low density lipoprotein-cholesterol (LDL-C), blood pressure, homocysteine, andpreventing platelet aggregation using a combination of a statin, three blood pressure lowering drugssuch as a thiazide, a β blocker, and an angiotensin converting enzyme (ACE) inhibitor each at halfstandard dose; folic acid; and aspirin-called as polypill- was estimated to reduce cardiovascularevents by ~80%. Essential fatty acids (EFAs) and their long-chain metabolites: γ-linolenic acid (GLA),dihomo-GLA (DGLA), arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid(DHA) and other products such as prostaglandins E1 (PGE1), prostacyclin (PGI2), PGI3, lipoxins(LXs), resolvins, protectins including neuroprotectin D1 (NPD1) prevent platelet aggregation, lowerblood pressure, have anti-arrhythmic action, reduce LDL-C, ameliorate the adverse actions ofhomocysteine, show anti-inflammatory actions, activate telomerase, and have cytoprotectiveproperties. Thus, EFAs and their metabolites show all the classic actions expected of the "polypill".Unlike the proposed "polypill", EFAs are endogenous molecules present in almost all tissues, haveno significant or few side effects, can be taken orally for long periods of time even by pregnantwomen, lactating mothers, and infants, children, and adults; and have been known to reduce theincidence cardiovascular diseases including stroke. In addition, various EFAs and their long-chainmetabolites not only enhance nitric oxide generation but also react with nitric oxide to yield theirrespective nitroalkene derivatives that produce vascular relaxation, inhibit neutrophil degranulationand superoxide formation, inhibit platelet activation, and possess PPAR-γ ligand activity and releaseNO, thus prevent platelet aggregation, thrombus formation, atherosclerosis, and cardiovasculardiseases. Based on these evidences, I propose that a rational combination of ω-3 and ω-6 fatty acidsand the co-factors that are necessary for their appropriate action/metabolism is as beneficial as thatof the combined use of a statin, thiazide, a β blocker, and an angiotensin converting enzyme (ACE)inhibitor, folic acid, and aspirin. Furthermore, appropriate combination of ω-3 and ω-6 fatty acidsmay even show additional benefits in the form of protection from depression, schizophrenia,Alzheimer's disease, and enhances cognitive function; and serve as endogenous anti-inflammatorymolecules; and could be administered from childhood for life long.

Published: 15 October 2008

Lipids in Health and Disease 2008, 7:37 doi:10.1186/1476-511X-7-37

Received: 30 September 2008Accepted: 15 October 2008

This article is available from: http://www.lipidworld.com/content/7/1/37

© 2008 Das; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Lipids in Health and Disease 2008, 7:37 http://www.lipidworld.com/content/7/1/37

IntroductionCardiovascular diseases (CVD) are responsible for signifi-cant morbidity and mortality throughout the world. Stud-ies revealed that smoking cessation, β-blockers, anti-platelet agents, angiotensin converting enzyme (ACE)inhibitors, and lipid lowering agents such as statins, eachreduce the risk of vascular events to a moderate but impor-tant degree [1-9]. In addition, observational studies sug-gested lower rates of fractures and dementia with statins,and lower rates of cataracts with anti-oxidant vitamins,though these observations need to be confirmed by ran-domised trials [9]. The results of the MRC/BHF-HPS studyled to the suggestion that using a combination of aspirin,β-blockers, statins, and ACE inhibitors could preventabout two-thirds to three-quarters of future vascularevents [10]. It was suggested that a combination pill(called as "polypill") consisting of atorvastatin 10 mg orsimvastatin 40 mg; three blood pressure lowering drugssuch as a thiazide, a β-blocker, and an ACE inhibitor, eachat half standard dose; folic acid 0.8 mg; and aspirin 75 mgcould reduce coronary heart disease (CHD) events by88% (95% confidence interval 84% to 91%) and strokeby 80% (71% to 87%), and if such a combination pill istaken from age 55 years of age, at least one third of peopletaking it, would on an average add about 11 years of lifefree from an CHD event or stroke [11].

Further support to the concept of polypill for the preven-tion of primary and secondary cardiovascular diseasesproposed by Wald and Law [11] is provided by the workof Hippisley-Cox and Coupland [12] who examined theindividual and combined effects of three of the polypillingredients-statins, aspirin, and blood pressure loweringdrugs. Their analysis of 11330 patients with CHD showedthat all cause mortality is lower in those taking two orthree drugs compared with those taking single agents.These findings are consistent with previous studies[13,14] that showed that a combination of two drugs-aspirin and statin-is superior to either drug alone in thesecondary prevention of CHD. However, it was also notedthat synergistic effects are seen with two, but not three orfour, drug combinations in secondary prevention of CHD.

But concerns have been raised about the adverse effects ofsuch a polypill. For instance, β blockers are unsuitable forsubjects with bronchial asthma, and some are intolerantto aspirin and develop significant gastrointestinal sideeffects. It may be necessary to closely monitor to detectserious adverse effects of statins, and renal failure due toACE inhibitors and angiotensin-II receptor antagonists.Furthermore, the efficacy of aspirin in men is established[15], but its efficacy in women is not certain [16]. A recentstudy showed that 2.5 mg of folic acid (the proposed poly-pill dosage is 0.8 mg/day) was not associated with a reduc-tion in stroke, coronary events, and death in patients who

previously had a cerebral infarction despite a moderatereduction of total homocysteine during the 2 years of fol-low-up [17]. Another major concern about the primaryprevention strategy of the "polypill" is related to the pos-sibility that it might lead to medicalising of the popula-tion since, it is highly cost effective to treat individuals athigh risk compared to those at lower risk that is muchmore expensive to treat in terms of gain in qualityadjusted life years. Hence, it will be worthwhile to look foralternatives to "polypill" that is less expensive, moreacceptable, less likely to cause side effects and does notlead to medicalising the population. I propose that arational combination of ω-3 and ω-6 fatty acids may, infact, be more beneficial compared to the "polypill" in theprimary and secondary prevention of cardiovascular dis-eases.

Metabolism of essential fatty acidsEssential fatty acids (EFAs) are essential for survival andthey cannot be synthesized in the body and hence, have tobe obtained in our diet and thus, are essential [18-21].There are two types of naturally occurring EFAs in thebody, the ω-6 series derived from linoleic acid (LA, 18:2)and the ω-3 series derived from α-linolenic acid (ALA,18:3). Both ω-6 and ω-3 series are metabolized by thesame set of enzymes to their respective long-chain metab-olites. While some of the functions of EFAs require theirconversion to eicosanoids and other products, in majorityof the instances the fatty acids themselves are active.

LA is converted to γ-linolenic acid (GLA, 18:3, n-6) by theaction of the enzyme Δ6 desaturase (d-6-d) and GLA iselongated to form dihomo-GLA (DGLA, 20:3, n-6), theprecursor of the 1 series of prostaglandins (PGs). DGLAcan also be converted to arachidonic acid (AA, 20:4, n-6)by the action of the enzyme Δ5 desaturase (d-5-d). AAforms the precursor of 2 series of prostaglandins, throm-boxanes and the 4 series of leukotrienes. ALA is convertedto eicosapentaenoic acid (EPA, 20:5, n-3) by d-6-d and d-5-d. EPA forms the precursor of the 3 series of prostaglan-dins and the 5 series of leukotrienes (see Figure 1 for themetabolism of EFAs). AA and EPA give rise to their respec-tive hydroxy acids, which, in turn, are converted to respec-tive leukotrienes (LTs). In addition, AA, EPA, and DHAform precursor to anti-inflammatory compounds lipox-ins, resolvins, and protectins (neuroprotectin D1 is onesuch compound derived from DHA) [22-27]. PGs, LTs,lipoxins (LXs), and resolvins are highly active, modulateinflammation, and are involved in several physiologicaland pathological processes [18]. In general, the term"EFAs" is used to refer to all unsaturated fatty acids: LA,GLA, DGLA, AA, ALA, EPA, and DHA; and the term poly-unsaturated fatty acids (PUFAs) refer to GLA, DGLA, AA,EPA, and DHA. Although the terms EFAs and PUFAs areused interchangeably, it should be understood that all




Metabolism of essential fatty acidsFigure 1Metabolism of essential fatty acids. Prostaglandins of 3 series are less pro-inflammatory compared to prostaglandins of 2 series. Resolvins are formed from both EPA and DHA and are known to have anti-inflammatory actions and participate in the resolution of inflammation. EPA can be converted to DHA. DHA can be retroconverted to EPA. It is estimated that about 30–40% of DHA can be retroconverted to EPA. The biochemical and/or clinical significance of this retroconversion of DHA to EPA are not known. ____ Indicates beneficial action in the form of increase in the synthesis, action, or remission of disease process. ____ Indicates decrease in the synthesis, action or enhancement of pathological process. ____ Indicates inhibition of HMG-CoA and ACE enzymes by PUFAs/EFAs.

1

Diet

-6 Cis-Linoleic acid (LA, 18:2)

- -Linoleic acid (ALA, 18:3)

-Linolenic acid (GLA, 18:3)

Dihomo- -Linolenic acid (DGLA, 20:3) (GLA, 18:3)

Arachidonic acid (AA, 20:4)

PGs of 2 ser ies PGA2, PGE2, PGF2 , PGI2, TXA2 LTB4, EETs, HETEs

Eicosapentaenoic acid (EPA, 20:5)

6 desaturase

5 desaturase

Statins Glitazones Folic acid,

Vit B12

Nitr ic Oxide

Docosahexaenoic acid (DHA, 22:6)

PGs of 3 ser ies PGA3, PGE3, PGF3 , PGI3, TXA3 LTB5, EETs, HETEs

HMG-CoA Reductase and ACE enzymes

Leukocytes Macrophages, Lymphocytes Platelets, Myocardial cells

MPO, Lp-PLA2 TNF- -6, CRP

LXs, Resolvins, Neuroprotectins, and Nitrolipids

Inflammation Atheroslcerosis Hyper tension

Insulin resistance Type 2 diabetes mellitus Coronary hear t disease

Stroke

urotransmission

Ageing Hyperglycemia

Saturated, Trans-fats, cholesterol

Insulin Calorie

restriction Zn, Mg2+,

Ca2+

Vitamin C, Zn, Ca2+

PGs of 1

series

Aspir in


EFAs are PUFAs but all PUFAs are not EFAs. Only LA andALA qualify to be EFAs; whereas GLA, DGLA, AA, EPA, andDHA are PUFAs. On the other hand, LA, GLA, DGLA, AA,ALA, EPA, and DHA are also called as LCPUFAs (long-chain polyunsaturated fatty acids). Many of the functionsof EFAs are also brought about by PUFAs and EFA-defi-ciency states can be corrected to a large extent by PUFAsthat suggests that PUFAs are "functional EFAs". Hence, ingeneral, many authors use the terms EFAs and PUFAsinterchangeably. This convention is followed in thepresent discussion.

Dietary sources of EFAsEFAs: LA and ALA are present in human diet in abundantamounts and hence, EFA-deficiency is uncommon. In cer-tain specific conditions such as total parenteral nutrition(TPN) and severe malabsorption occasionally EFA defi-ciency could be seen. The present TPN solutions containadequate amounts of EFAs. The manifestations of EFAdeficiency include: dry and scaly skin, hepatospleenome-galy, immunodeficiency, inappropriate water lossthrough the skin, dehydration, scalp dermatitis, alopecia,and depigmentation of hair. EFAs are widely distributedin normal human diet. The main dietary sources of EFAsare as follows.

Human breast milk is rich in all types of PUFAs [18] thatexplains why breast-fed children are healthier comparedto bottle-fed. LA and ALA are present in significantamounts in dairy products, organ meats such as liver, andmany vegetable oils such as sunflower, safflower, cornand soy. GLA is present in evening primrose oil at concen-trations of 7–14% of total fatty acids; in borage seed oil itis 20–27%; and in black currant seed oil at 15–20%. GLAis also found in some fungal sources [18]. DGLA is foundin liver, testes, adrenals, and kidneys. AA is present inmeat, egg yolks, some seaweeds, and some shrimps. Aver-age daily intake of AA is estimated to be ~100–200 mg/day that accounts for the total daily production of variousPGs. EPA and DHA are present mainly in marine fish.Cow's milk contains very small amounts of GLA, DGLAand AA.

EFAs/PUFAs are unstable due to the presence of 2 or moredouble bonds in their structure. Substantial loss of EFAs/PUFAs occurs during food processing and hydrogenationof oils. Exposure to high temperatures and hydrogenationprocess causes denaturation of EFAs/PUFAs and their con-version to trans-fats that are harmful to the body [18].Human diet was rich in ω-3 fatty acids in the earlyhumans. But with the progress in industrialization anddevelopment of fast foods, the content of ω-3 fatty acidsin human diet dwindled, whereas that of ω-6 fatty acidsincreased. The ratio of ω-3 to ω-6 fatty acids in the diet ofearly humans was >1, whereas this ratio is now about 10:1

to 20–25:1. It is recommended that the ratio between ω-3to ω-6 fatty acids in the diet should be about 1 or >1 andpreferably 2–3:1. This fall in the intake of ω-3 fatty acids,especially EPA and DHA in the last 50 years is believed tobe responsible for the increasing incidence of atheroscle-rosis, CHD, hypertension, metabolic syndrome X, obesity,collagen vascular diseases and possibly, cancer. This issupported by the observation that increasing dietary α-linolenate/linoleate balance affected the ω-3/ω-6 ratio ofbrain phospholipid acyl chains and produced changes ingeneral behavior as well as changes in sensitivities todrugs known to affect behavior, influenced LTs formationin polymorpho-nuclear leukocytes from AA and EPA andrelease of histamine from mast cells that could alter theseverity of allergic and inflammatory responses. Thisincrease in ω-3 fatty acids also resulted in an increasedmean survival time of SHR-SP (spontaneously hyperten-sive-stroke prone) rats by lowering blood pressure andplatelet aggregability, produced significant changes inNa+-K+-ATPase activity, and altered collagen-inducedplatelet aggregation and serotonin release in experimentalanimals. These results suggest that enhanced intake of ω-3 fatty acids is of significant benefit in various diseases.

Factors influencing the metabolism of EFAsDietary LA and ALA are metabolized by the same set of Δ6

and Δ5 desaturases and elongases to their respectivemetabolites (see Figure 1). These 2 fatty acids competewith one another for the same set of enzymes and Δ6 andΔ5 desaturases prefer ω-3 to ω-6. Oleic acid (OA, ω-9) thatis not an EFA is also metabolized by the same desaturases.But, in view of the preference of these enzymes to LA andALA, under normal physiological conditions, the metabo-lites of ω-9 are formed only in trivial amounts. Hence,presence of significant amounts of 20:3 ω-9, a metaboliteof OA, in the cells and plasma indicates EFA deficiencythat is utilized to detect the presence of EFA deficiency inpatients, experimental animals and in vitro studies.

Of several factors that influence the activities of desatu-rases and elongases [18], saturated fats, cholesterol, trans-fatty acids, alcohol, adrenaline, and glucocorticoidsinhibit Δ6 and Δ5 desaturases. Pyridoxine, zinc, and mag-nesium are necessary co-factors for normal Δ6 desaturaseactivity. Insulin activates Δ6 desaturase whereas diabeticshave reduced Δ6 desaturase activity. The activity of Δ6

desaturase falls with age. Oncogenic viruses and radiationinhibit Δ6 desaturase. Total fasting, protein deficiency,glucose rich diets reduce the activity of Δ6 desaturase. Afat- free diet and partial caloric restriction enhances Δ6

desaturase. Activities of Δ6 and Δ5 desaturases aredecreased in diabetes mellitus, hypertension, hyperlipi-demia, and metabolic syndrome X. Trans-fats, saturatedfatty acids, and cholesterol interfere with EFA metabolismand promote inflammation, atherosclerosis and coronary



heart disease (CHD) [18]. This implies that trans-fats, sat-urated fats, and cholesterol have pro-inflammatoryactions whereas EFAs and PUFAs possess anti-inflamma-tory properties. This explains why trans-fats, saturatedfats, and cholesterol are pro-atherogenic, whereas EFAs/PUFAs, especially ω-3 fatty acids are anti-atherogenic. Theability of trans-fats, saturated fats, and cholesterol to inter-fere with the formation of AA, EPA, and DHA from dietaryLA and ALA could lead to decreased formation of LXs,resolvins, PGI2 (prostacyclin), PGI3, and other beneficialeicosanoids that prevent platelet aggregation, leukocytechemotaxis and activation. LXs and resolvins decrease theformation of pro-inflammatory cytokines, and producevasodilatation, events that prevent or arrest atheroslcero-sis. In contrast, trans-fats, saturated fats, and cholesterolmay directly activate leukocytes, induce the generation offree radicals and enhance the production and release ofpro-inflammatory cytokines that facilitate atherosclerosis[18]. Trans-fats, saturated fats, and cholesterol directlyactivate leukocytes and macrophages to induce them toproduce free radicals and pro-inflammatory cytokines:

IL-6, TNF-α, IL-1, IL-2, and MIF (macrophage migrationinhibitory factor). This action of trans-fats, saturated fats,and cholesterol is in addition to their ability to suppressmetabolism of EFAs to their respective long-chain metab-olites. It is possible that trans-fats, saturated fats, and cho-lesterol may also have the ability to inhibit the formationof LXs, resolvins, PGI2, and PGI3. These studies suggestthat EFAs, especially EPA and DHA are cytoprotective toendothelial cells, whereas trans-fats, saturated fats, andcholesterol produce endothelial dysfunction. AA, EPA,and DHA augment nitric oxide generation from endothe-lial cells [18] and thus help in the prevention of endothe-lial dysfunction. In contrast, trans-fats, saturated fats, andcholesterol produce endothelial dysfunction and thus,inhibit eNO production. Furthermore, NO quenchessuperoxide anion and thus, prevents the cytotoxic actionof superoxide anion and protects endothelial cells fromfree radical-induced damage. This implies that forendothelial cells to be healthy they need adequateamounts of AA, EPA, and DHA so that they can generatephysiological amounts of eNO not only to prevent patho-logical platelet aggregation and atherosclerosis but also toprotect themselves from the cytotoxic actions of free radi-cals.

Furthermore, NO reacts with PUFAs to yield their respec-tive nitroalkene derivatives that can be detected in plasma.These nitroalkene derivatives, termed as nitrolipids, pro-duce vascular relaxation, inhibit neutrophil degranulationand superoxide formation, and inhibit platelet activation,and show anti-atherosclerotic properties [18]. Thus, thereappears to be a close interaction between EFAs and theirproducts and trans-fats, saturated fats, and cholesterol

with regard to the ability of endothelial cells to producePGI2, PGI3, NO, and other anti-atheroslcerotic and bene-ficial molecules.

Actions of PUFAs that qualify them to be the endogenous HMG-CoA reductase and ACE enzyme inhibitors, anti-arrhythmic, anti-hypertensive, anti-atherosclerotic, anti-inflammatory, cytoprotective, and cardioprotective moleculesAction on HMG-CoA reductasePUFAs are potent inhibitors of HMG-CoA reductaseenzyme and similar to statins are useful in the treatmentof hyperlipidemias [28-33]. Statins enhance plasma AAlevels and decrease the ratio of EPA to AA significantly[29,30], and enhance the formation of prostacyclin(PGI2) [32]. In fact, statins and PUFAs have many overlapactions such as inhibition of IL-6 and TNF-α productionand NF-κB activation, increasing the synthesis of endothe-lial nitric oxide (eNO); and both are anti-inflammatory innature [18,33-37]. In addition, a close interaction existsbetween NO and COX enzymes attesting to the fact thatstatins, PUFAs, and NO have positive and negative influ-ences among themselves [38]. Both statins and PUFAs areuseful in atherosclerosis, coronary heart disease, oste-oporosis, stroke, Alzheimer's disease, and inflammatoryconditions such as lupus [18,39-52]. These evidences sug-gest that PUFAs mediate many, if not all, actions of statins[33] and this could be one mechanism by which theylower cholesterol levels. Recent studies revealed that stat-ins augment concentrations of LXs in the heart [53,54]lending support to this concept. Furthermore, when acombination of statins and PUFAs are given together asynergistic beneficial effect was seen in patients with com-bined hyperlipemia [55-58]. But statins cannot be givenduring pregnancy, whereas PUFAs have been recom-mended during pregnancy, lactation and infancy toimprove brain growth and development, and cognitivefunction [18-21], [59-67], though some studies did notshow improvement in cognition [68,69]. Nevertheless,these studies reported that supplementation of PUFAs topregnant and lactating women and infants is safe andwithout any side effects.

PUFAs modulate renin formation, ACE activity and endothelial nitric oxide generationAngiotensin converting enzyme (ACE) inhibitors are use-ful to lower blood pressure [11]. Renin, a proteolyticenzyme, is produced and stored in the granules of the jux-taglomerular cells in the kidney. Renin acts on angi-otensinogen (a circulating α2 globulin made in the liver)to form the decapeptide angiotensin-I (Ang-I). Ang-I istransformed by ACE to angiotensin-II (Ang-II). Ang-IIcontrols blood pressure and regulates body fluid volumeby modulating renin-angiotensin-aldosterone system.



ACE is present in the uterus, placenta, vascular tissue,heart, brain, adrenal cortex and kidney, leukocytes, alveo-lar macrophages, peripheral monocytes, neuronal cellsand epididymal cells [70]. Angiotensin receptors are AT1and AT2. AT1 exists as two subtypes α and β. Actions ofAng-II are mediated by the AT1 receptor. Angiotensinases,present in several tissues, destroy Ang-II (half-life approx-imately 1 minute), while the half-life of renin is about 10to 20 minutes. In addition to circulating renin-angi-otensin, many tissues have a local renin-angiotensin sys-tem and thus, have the ability to produce Ang-II. Locallygenerated Ang-II is involved in the modulation of growthand function of many tissues including vascular smoothmuscle.

Linseed oil (which contains approximately: 19% oleicacid, 24% LA, and 47% ALA) fed experimental animalsshowed significantly lower renal venous renin secretionrates relative to the saturated fat-fed control group. Die-tary enrichment with 20 energy % PUFA lowered reninsecretion by a prostaglandin-independent mechanismthat might have contributed to the lower blood pressuresobserved compared with the saturated fat-fed controlgroup [71]. Dietary supplementation with 3 gm/day of LAand 32 mg/day of GLA to pregnant and non-pregnant sub-jects showed that the diastolic pressor response to angi-otensin-II was significantly less in the pregnant subjectscompared to the non-pregnant subjects, suggesting thatPUFAs modulate vascular tissue responses to angiotensin-II. This decreased response to angiotensin-II could be dueto increased formation of PGE1 and PGI2 in fatty acid sup-plemented pregnant subjects [72,73]. In a model ofhypertension induced by continuous infusion of angi-otensin-II in the rat, subcutaneous administration of LAand EPA and DHA were found to be equally potent inreducing, by half, the rise in systolic blood pressureinduced by angiotensin-II and these anti-hypertensiveeffects were not accompanied by any changes in the renalsynthesis of PGI2 or PGE2. Furthermore, indomethacin, apotent inhibitor of PGI2 but not of PGE2 synthesis couldonly partially neutralize the anti-hypertensive effects of LAand EPA/DHA, emphasizing that the anti-hypertensiveeffects are independent of the PG system [74]. This idea isreinforced by the observation that LA and AA inhibitrenin, and thus, the overall activity of the renin-angi-otensin-aldosterone cascade could be modified by altera-tions of plasma fatty acid concentrations [75,76], thoughin two-kidney, one-dip hypertensive animals, pretreat-ment with indomethacin did not alter hypotensiveresponse to LA despite the fact that LA infusion loweredblood pressure in high renin but not in low renin statesand the reduction in blood pressure was not related toinhibition circulating renin or to alterations of endog-enous PG biosynthesis [77]. These evidences suggest thatPUFAs have modulatory influence on renin secretion and

action and yet times independent of both renin secretionand PG formation. Since the anti-hypertensive actions ofPUFAs seem to be independent of formation of PGs, it islikely that fatty acids themselves are able to bring aboutthis action and/or converted to form lipoxins, resolvinsand protectins that have anti-inflammatory action andalso possibly, anti-hypertensive action. But this proposalneeds to be verified and confirmed. Yet another possibil-ity is that the PUFAs supplemented are incorporated intothe cell membrane phospholipid fraction that is able toenhance the synthesis of eNO that is a potent vasodilatorand platelet anti-aggregator.

Previously, I showed that PUFAs inhibit leukocyte ACEactivity [78]. Of all the fatty acids tested, EPA was the mosteffective (EPA > ALA > DHA > GLA > LA > AA), whereasAA was the least effective when their ability to inhibitpurified ACE activity was tested. DHA and EPA were themost effective fatty acids in inhibiting the leukocyte ACEactivity (EPA > DHA > ALA = AA > LA > GLA). On theother hand, PGs (PGE1, PGE2, PGI2 and PGF2α), and freeradicals: superoxide anion, hydrogen peroxide, andhydroxyl radical showed marginal (~20%) inhibitoryaction on ACE activity. In contrast, NO (nitric oxide)showed powerful inhibitory action on ACE activity [78],whereas PUFAs enhanced endothelial nitric oxide (eNO)generation [36,47,79]. The effects of PUFAs on ACE activ-ity, and NO generation and the inability of free radicalsand PGs to suppress ACE activity are interesting sincethere is a close interaction between platelets, leukocytesand endothelial cells that may have relevance to theirinvolvement in CVD (cardiovascular diseases). Forinstance, under normal conditions, endothelial cells pro-duce adequate amounts of PGE1 from DGLA; PGI2 fromAA; LXs and resolvins from AA, EPA and DHA; and NOfrom L-arginine such that the pro-inflammatory and pro-atherosclerotic events such as hemodynamic forces,hyperlipidemia, hypertension, smoking are successfullyabrogated. These factors induce the expression of pro-inflammatory genes that initiate and accelerate athero-sclerosis at the points of shear stress, enhance infiltrationof intima by leukocytes and macrophages, cause low-levelactivation of NF-κB and elevated expression of VCAM-1and ICAM-1, IL-1, IL-6, MCP-1, as well as antioxidantgenes glutathione peroxidase and glutathione-S- trans-ferase 2, and pro-inflammatory eicosanoids such as TXA2,PGE2, PGF2α, LTs, and other PGs, TXs, and LTs, andincreased production and release of free radicals and UCP(uncoupling proteins) expression occurs in endothelialcells, platelets, and leukocytes in atherosclerosis-suscepti-ble regions, and endothelial cells themselves may showchanges in cell shape and proliferation. These events canbe prevented and atherosclerosis process and the onset ofCVD can be arrested if the production of PGE1, PGI2,PGI3, LXs, resolvins, NO, and anti-inflammatory



cytokines such as IL-4, IL-10, TGF-β by endothelial cells isadequate, provided there are adequate stores of respectiveprecursors of various PUFAs and L-arginine and theirrespective enzymes. This suggests that under physiologicalconditions a delicate balance is maintained between pro-and anti-inflammatory and pro and anti-atheroscleroticfactors and when this balance is tilted more towards theformer atherosclerosis and CVD occurs (reviewed in [80]).

In addition, these results suggest that when tissue concen-trations of PUFAs are low, the activity of ACE will be highresulting in increased formation of angiotensin-II and asimultaneous decrease in eNO. In this context, it is impor-tant to note that transgenic rats overexpressing humanrenin and angiotensinogen genes (dTGR) develop hyper-tension, inflammation, and renal failure, and showed spe-cific renal P450-dependent AA metabolism changes thatled to decreased formation epoxy-eicosatrienoic acids(5,6-, 8,9-, 11,12- and 14,15-EETs) and hydroxyeicosa-tetraenoic acids (19- and 20-HETEs). Both EETs andHETEs inhibit IL-6 and TNF-α-induced activation of NF-κB and prevent vascular inflammation [81], suggestingthat AA and other PUFAs not only regulate ACE activityand Ang-II levels in the tissues but also possess anti-inflammatory properties by generating anti-inflammatorymetabolites.

AA, EPA, and DHA are converted in the presence of aspirinto epi-lipoxins, lipoxins, and resolvins that possess potentanti-inflammatory actions (reviewed in [18]). Epi-lipox-ins enhance the formation of eNO [18,21,22]. NO blocksthe interaction between leukocytes and the vascularendothelium and also stimulates the formation of PGI2, apotent vasodilator and platelet anti-aggregator, from AA[82]. This suggests that the beneficial actions of aspirincould be attributed not only to its ability to enhance theformation of PGI2 and suppress the synthesis of TXA2 butalso to the formation of epi-lipoxins and eNO [20,21,82].Thus, PUFAs regulate renin formation and action, inhibitangiotensin-II formation by its action on ACE activity,enhance eNO formation, and form precursors to benefi-cial biologically active molecules such as PGE1 (fromDGLA), PGI2 (from AA), PGI3 (from EPA), lipoxins (fromAA, EPA, and DHA), resolvins (from AA, EPA and DHA),protectins (from DHA), and 5,6-, 8,9-, 11,12- and 14,15-EETs and hydroxyeicosa-tetraenoic acids (19- and 20-HETEs) (from AA), and thus serve as endogenous regula-tors of vascular tone, platelet aggregation, and blood pres-sure.

Effects on platelets and other hemostatic indicesAspirin is effective in the prevention and treatment ofacute myocardial infarction (AMI) and in the secondaryprevention of CVD [83], though the efficacy of aspirin inwomen is not certain [16]. One of the important constit-

uents of the "polypill" is aspirin (75 mg). Studies revealedthat low dose aspirin not only reduced risk of heart dis-ease, but also reduced the incidence of lung, colon, andbreast cancer [84]. Aspirin inhibits nuclear factor NF-κBtranscription, blocks prostaglandin (PG) and thrombox-ane (TX) synthesis (TXA > PGI2). Aspirin does not inhibitthe production of proinflammatory mediators such asleukotrienes (LTs). Although blockage of PGs and TXsaccounts for many of aspirin's pharmacologic properties,recent studies revealed that aspirin evokes the formationof 15-epi-LXA4 by the acetylated PGHS-2 [prostaglandinG/H synthase (cyclooxygenase)] and 5-lipoxygenaseenzymes as a result of endothelial cell-leukocyte interac-tions [85]. LXA4 inhibits polymorphonuclear leukocytetransmigration, modulate adhesion to endothelial cells,and inhibit chemotaxis of PMN and eosinophils. Thus,many beneficial actions of aspirin could be attributed tothe formation of LXA4. In this context, it is noteworthythat AA, EPA, and DHA when used in appropriate concen-trations and ratio can reproduce many of the beneficialactions of aspirin.

Both EPA and DHA, when given orally, are rapidly incor-porated into platelets and compete with AA for the 2-acylposition of membrane phospholipid and as substrate forthe cyclo-oxygenase (CO) and lipoxygenase (LO)enzymes. As a result, when stimulated, such platelets pro-duce less amounts of TXA2 and more of TXA3 that is lesspotent in inducing platelet aggregation and thrombosis[86]. Increased intake of fish oil, a rich source of EPA andDHA, produces a lower platelet count, less platelet aggre-gation, a longer bleeding time, higher urinary PGI2 metab-olites, and lower concentrations of thromboxanemetabolites compared to those who were on Western diet[87,88], effects that are similar to those of low-dose aspi-rin and qualify ω-3 EPA and DHA to be termed as an"endogenous aspirin". In general, though EPA and DHAdo not have a very significant effect on blood lipids(except to lower plasma triglycerides and VLDL with nosignificant action on HDL-C levels), fibrinolysis and onthe activity of plasminogen activity inhibitor-type-1 (PAI-1), still are effective in preventing overall mortality fromCVD [18,21,80,89-94].

It is believed that production of pro-inflammatory eicosa-noids from AA and/or decreased synthesis of anti-inflam-matory and beneficial eicosanoids from EPA/DHA couldpredispose an individual to cardiovascular risk and stroke.Thus, it is thought that products of AA such as TXA2, PGEs,PGFαs, and LTs contribute or initiate the process of athero-sclerosis in coronary arteries and cause CHD. In contrast,it has been proposed that beneficial products or lessharmful products formed from EPA and DHA are lesslikely to cause atherosclerosis and CHD or even regressatheroma and prevent CHD. Although this is an attractive



hypothesis, hard data in support of this proposal are notforthcoming. In this context, it should be noted that notall products of AA are harmful. For instance, PGI2 formedfrom AA is a potent vasodilator and platelet anti-aggrega-tor and prevents atherosclerosis and has anti-arrhythmicaction [95].

Similarly, DGLA, another ω-6 fatty acid that is the precur-sor of AA, gives rise to PGE1, which is a vasodilator, plate-let anti-aggregator and anti-arrhythmic molecule [96,97].These data emphasize the complexities involved in mak-ing generalizations about ascribing negative role to ω-6fatty acids and their products in CVD. Furthermore, AAforms precursor to LXs and resolvins (See Figure 1) thathave beneficial actions in resolving inflammation. Inaddition, Harris et al [98] noted that pooling of data fromcase-control or prospective cohort studies showed thatnone of the individual fatty acids computed across data-sets were significantly different between cases and con-trols. EPA was 8.2% lower (p = 0.06), DHA was 8% lowerin cases compared with controls, whereas DPA (docosap-entaenoic acid) was virtually identical in both controlsand CHD patients. In contrast and contrary to expecta-tions, AA was 8.5% lower in cases compared to controls.The unexpected finding that AA concentrations werelower in patients with CHD suggests that it is the defi-ciency of AA rather than its excess that predisposes toCHD events. In addition, data from the Health Profes-sionals' Follow-up Study [99] revealed that while there isan inverse relationship between ω-3 fatty acid intake andfuture risk for CHD, higher intakes of ω-6 fatty acids didnot diminish the beneficial effects of ω-3 fatty acids sug-gesting that the absolute intakes of the ω-3 fatty acids aremore important than the ratio between ω-3 fatty acids andω-6 fatty acids. In this context, the interaction(s) betweenω-3 and ω-6 fatty acids are significant.

Interaction(s) between ω-3 and ω-6 fatty acids and its relevance to CHD/CVDIn a case control study of new angina pectoris and firstacute myocardial infarction, a progressive inverse relationbetween adipose tissue LA and the estimated relative riskof CHD was noted [100]. Wood, et al [101] observed thatlow concentrations of DGLA in adipose tissue showed amore significant relation to new CHD than did LA. In anextension of this study, it was noted that there is a progres-sive inverse relations between adipose LA and platelet-membrane EPA and the estimated relative risk of anginapectoris. These relations were statistically independent ofeach other and traditional CHD risk factors [100-102].

Luostarinen, et al [103] noted that the percentage of pal-mitic acid and LA were significantly higher and the per-centage of AA and of all the other major PUFAs, both ω-3and ω-6, was significantly lower in the total phospholipid

fraction of human coronary arteries of those who had sud-den cardiac death due to CHD. Felton, et al [104] reportedthat the concentrations of all fatty acids were increased atthe edge of disrupted plaques compared with the center,but as a proportion of total fatty acids, ω-6 were lower.These results suggest that ω-6 fatty acids have a significantrole in CHD and it is likely that some of the inconsistentresults obtained in some studies with EPA and DHA couldbe attributed to inadequate provision or utilization of ω-6 fatty acids, DGLA and AA. It is possible that there is aclose interaction between ω-3 and ω-6 fatty acids, whichcould influence one's susceptibility or resistance to CHD.In this context, it is interesting to note that EPA/DHAreadily get incorporated into the atheromatous plaque,and patients treated with fish oil had more thick fibrouscaps and no signs of inflammation compared withplaques in patients in the control and sunflower oilgroups. Furthermore, the number of macrophages inplaques from patients receiving fish oil was lower than inthe other two groups, suggesting that atheroscleroticplaques readily incorporate ω-3 PUFAs from fish-oil sup-plementation, inducing changes that can enhance stabil-ity of atherosclerotic plaques [105].

Studies revealed that ω-3 and ω-6 fatty acids interact witheach other in such a way that one potentiates the metabo-lism of the other. For instance, in perfused vascular tissue,DGLA increases the conversion of EPA to PGI3, a potentvasodilator and platelet anti-aggregator [106], whereas AAaugmented the conversion of EPA to PGI3 in the tissues[107-109]. EPA inhibits the activity of the enzyme Δ5

desaturase that results in an increase in the concentrationsof DGLA in the tissues (especially in the endothelial cells).This increase in tissue levels of DGLA could enhance theformation of PGE1, a vasodilator and platelet anti-aggre-gator (see Figure 1). Thus, EPA can indirectly enhance theformation of PGE1. Furthermore, even the beneficialaction of statins (HMG-CoA reductase inhibitors) and gli-tazones (PPARs agonists) seem to be mediated by EFAsand their metabolites such as LXs, resolvins, and neuro-protectins [28-33], [110-114], which are potent anti-inflammatory molecules [18,21,115-117]. Studies didsuggest that ω-3 fatty acids decreased the levels of pro-inflammatory cytokines, and enhance that of IL-10, ananti-inflammatory cytokine [118-120]. This close interac-tion between ω-3 and ω-6 fatty acids and their ability tomodify inflammatory markers, production of PGI2, PGE1,PGI3, LXs, resolvins, neuroprotectins, NO, nitrolipids,and the action of statins and glitazones on EFA metabo-lism and NO explains the relationship between variousfatty acids and CHD and stroke (Figure 1).

PUFAs in renal functionOne of the components suggested to be included in the"polypill" is a thiazide, a diuretic and an anti-hypertensive



drug. If PUFAs are to be considered to function as anendogenous "polypill", then they (PUFAs) should showbeneficial actions on renal function.

Healthy volunteers given EPA (3.9 gm) and DHA (2.4 gm)per day for 6 weeks showed significant increase in renalplasma flow, glomerular filtration rate, decrease in renalvascular resistance, and an increase in excretion of PGE3with no change in blood pressure and heart rate [121].Diet rich in evening primrose oil (a rich source of GLA andLA) and safflower oil decreased proteinuria, glomerularsclerosis, and tubular abnormalities in diabetic rats, andshowed increased ratio of renal cortical production of 6-keto-PGF1α (a metabolite of PGI2) to TXB2 with no signif-icant changes in plasma lipid composition. In contrast,fish oil feeding decreased plasma lipids and lowered 6-keto-PGF1α/TXB2 ratio without any effect on renal diseasein diabetic rats [122]. Singer et al [123] observed thatspontaneously hypertensive rats had significantly lowersystolic blood pressure when fed fish oil (EPA and DHA),evening primrose oil (a rich source of GLA), and fish oil +evening primrose oil, suggesting that a combination ofGLA, EPA, and DHA produces optimal beneficial actionswith regard to renal indices and blood pressure. Vaskonenet al [124] reported that fish oil prevented rise in bloodpressure induced by high-salt diet in stroke-prone sponta-neously hypertensive rats. This beneficial effect on bloodpressure was associated with a decrease in TXB2 formationby 75% and an increase in plasma and renal ω-3 fatty acidcontent.

Furthermore, EPA/DHA suppressed mesangial cell prolif-eration, arrested progression of IgA nephropathy, andprotected against cyclosporine-induced renal damage[125-127]. These results suggest availability of optimalamounts of GLA and EPA/DHA is necessary to reduceblood pressure and preserve renal function in diabetic andhypertensive rats. Studies performed with 5/6 renal abla-tion rat model that developed hypertension, albuminuria,and a decline in glomerular filtration rate had signifi-cantly less glomerulosclerosis and dyslipidemia whensupplemented with fish oil and flax seed oil (rich in ALA)compared with the control group at 10 and 20 wk post-surgery [128,129]. Thus, PUFAs may show actions similarto those observed with conventional, synthetic diuretics.These beneficial actions of PUFAs can be attributed to theformation of beneficial PGA, PGE3, PGI2, PGI3, andrecently identified resolvins and protectins and decreasein the production of TXA2 and LTs [130]. It is interestingthat diuretic furosemide enhances endothelial synthesisand release of bradykinin and related kinins that, in turn,stimulates endothelial PGI2 formation via B2 kinin recep-tor activation [131] and COX-2 derived PGs interact withthe renin-angiotensin system to regulate renal function[132].

PUFAs and parasympathetic nervous systemPrevious studies showed that multiple-blood pressurelowering drugs have additive effects, which led Wald andLaw to suggest that a β-blocker need to be added to the"polypill" composition. This is in addition to the presenceof a diuretic (such as a thiazide) and an ACE-inhibitor[11]. Autonomic function is an important factor that reg-ulates heart rate, blood pressure and cardiac rhythm.Hence, it is expected that addition of β-blocker to thecomposition of "polypill" will reduce sympathetic tone,blood pressure and heart rate.

Autonomic function is assessed by the measurement ofheart rate variability (HRV) and the evaluation of barore-flex sensitivity (BRS). HRV reflects the physiological levelsof tonic autonomic regulation, whereas BRS indicates thecapacity of reflex autonomic regulation. Both low HRVand low BRS are associated with increased cardiovascularrisk. Vagal stimulation by a release of acetylcholine (ACh)and adrenergic stimulation mediated by norepinephrineand epinephrine regulate the autonomic function andthus the variations in HRV and BRS. Several studiesrevealed that ω-3 fatty acids reduce the risk of suddendeath by preventing life-threatening cardiac arrhythmiasand by significantly increasing HRV [133]. Furthermore, adirect positive correlation was noted between the contentof DHA in cell membranes and HRV index suggesting ananti-arrhythmic effect of ω-3 fatty acids [134]. Sinceincreased parasympathetic tone is responsible for increasein ventricular fibrillation threshold and protects againstventricular arrhythmias, it is likely that EPA/DHA supple-mentation enhances parasympathetic tone. This is sup-ported b the observation that EPA/DHA supplementationincreases hippocampal ACh levels, the principal neuro-transmitter of parasympathetic nerves [135]. Hence, it islikely that EPA/DHA supplementation increases the brainACh levels leading to an increase in the parasympathetictone and so an increase in HRV and protection from ven-tricular arrhythmias. Similar to EPA/DHA, AA also aug-ments ACh release [136,137], and thus, PUFAs enhanceparasympathetic tone resulting in an increase HRV andprevention of ventricular arrhythmias.

Vagus nerve stimulation also inhibits TNF synthesis inliver and ACh significantly attenuated the release of pro-inflammatory cytokines: TNF-α, IL-16, IL-1β, and IL-18but not anti-inflammatory cytokine IL-10 by stimulatedmacrophages in vitro and in vivo [138-140]. Thus, onemechanism by which PUFAs suppress inflammationcould be by augmenting the release of ACh and enhancingthe parasympathetic tone.

Since, normally a balance is maintained between para-sympathetic and sympathetic tones, it is reasonable tosuggest that whenever parasympathetic tone (vagal tone)



is enhanced sympathetic tone is reduced (akin to blockingof β-receptors as it occurs in instances of use of β-block-ers). Thus, indirectly PUFAs may function like β-blockers.

Folic acid and PUFAsHyperhomocysteinemia is a risk factor for cardiovasculardiseases, and it may interact with hypertension and anunfavorable cholesterol profile to alter the risk of CVD.Hence, folic acid (0.8 mg/day) has been added as a com-ponent of the "polypill". It is important to note that folicacid increases concentrations of ω-3 PUFAs which couldreduce the risk of thrombosis and CVD [141-143]. It wasobserved that some of the adverse effects induced by folicacid deficiency could be overcome by supplementing withω-3 EPA and DHA [144], and in folic acid deficiency statesthe plasma and brain concentrations of PUFAs aredecreased [145]. These results imply that some, if not all,actions of folic acid are mediated by PUFAs.

Thus, PUFAs, when given in appropriate dose and combi-nation (containing EPA, DHA and possibly, GLA, DGLAand AA) show all the qualities of the suggested "polypill",viz., aspirin-like action, inhibit the activities of HMG-CoAand ACE enzymes, possess diuretic and anti-hypertensiveactions, and indirectly show β-blocker-like action. Inaddition to these useful actions, PUFAs also have otherbeneficial actions as described below.

PUFAs inhibit cholesteryl ester transfer protein (CETP) activityHDL-cholesterol (high-density lipoprotein- cholesterol,HDL-C) is an independent risk factor for CHD. Higherplasma HDL-C is associated with a decreased incidence ofCHD [146] that led to the suggestion that therapeuticstrategies that raise HDL-C could be of benefit in prevent-ing CHD by increasing the movement of cholesterol fromthe periphery back to the liver (the so-called reverse cho-lesterol transport or RCT pathway).

CETP is a hydrophobic plasma glycoprotein, mainly syn-thesized in the liver, possessing the unique ability to facil-itate the transfer of cholesteryl ester (CE). CETP circulatesin the blood, bound predominantly to HDL. CETP medi-ates the transfer of cholesteryl esters from HDL to VLDLand LDL in exchange for triglycerides and promotes thetransformation of HDL2 to HDL3, an action that couldpromote reverse cholesterol transport. CETP inhibitionproduces an increase in HDL by markedly delaying catab-olism of apoA-I and A-II [147], an action that increasesreverse cholesterol transport. These actions suggest thatCETP inhibition could prevent atherosclerosis and pre-vent CHD [148-150].

In healthy, normolipidemic men, it was observed thatlipid-lowering diet rich in monounsaturated fatty acid

(oleic acid) decreased CETP concentrations to a signifi-cant degree [151]. In HepG2 cells, 0.5 mM of AA, EPA,and DHA reduced the levels of CETP mRNA by more than50% of the control levels with a corresponding significantdecrease in the CETP mass [152]. A significant negativecorrelation was found between plasma CETP activity andmonounsaturated fatty acid content of plasma phosphol-ipids or free PUFAs including ω-3 fatty acids, suggestingthat PUFAs suppress CETP activity [153].

Torcetrapib, a small molecule inhibitor of CETP, is veryeffective at raising HDL-C and apolipoprotein A-I anddecreasing levels of LDL-C and apolipoprotein-B-100 andalso showed favourable effects on increasing the size ofHDL-and LDL particles. In patients with familial hyperc-holesterolemia, torcetrapib with atorvastatin as comparedwith atorvastatin alone did not result in reduction of pro-gression of atheroslcerosis as measured by carotid arterial-wall thickness despite a significant increase in HDL-C lev-els and decrease in levels of LDL-C and triglycerides. Infact, it was observed that administration of torcetrapibwith atorvastatin was associated with progression ofatherosclerosis [154], and an increase in blood pressurewith no significant decrease in the progression of coro-nary atherosclerosis [155]. These results with torcetrapib +atorvastatin suggest that simultaneous inhibition of CETPand HMG-CoA reductase enzyme leads to an elevation ofplasma HDL-C, and decrease in LDL-C and triglyceridesand cholesterol but it does not arrest progression ofatherosclerosis. In contrast, PUFAs, especially ω-EPA andDHA, not only inhibit CETP and HMG-CoA reductaseenzyme, lower plasma triglycerides, cholesterol, and LDL-C with little or no change in HDL-C but also are effectivein arresting atherosclerosis and preventing CHD [156-165]. In contrast to the results with torcetrapib + atorvas-tatin, Yokoyama et al [166] reported that a combinationof ethyl EPA + 10 mg of pravstatin or 5 mg of simvastatinprevented major coronary events and especially non-fatalcoronary events in Japanese hypercholesterolemicpatients with a mean period of follow up of 4.6 years. It isinteresting to note that the benefits were in addition tostatin treatment, and fish oil was found to be safe and welltolerated. These results once again confirm that EPA andDHA are of benefit in the prevention and treatment of car-diovascular diseases. Thus, PUFAs appear to be superior toCETP and statins in the prevention of CVD despite the factthat they do not necessarily increase plasma HDL-C levels.

EPA, DHA, and PGI2 function as endogenous anti-arrhythmic moleculesThere is reasonable evidence to suggest that EPA/DHA andPGI2 have anti-arrhythmogenic effects. Various PUFAsand PGs are present in the heart including SA (sinoatrial)node [167,168]. I showed that PGE1, PGE2, PGI2, andTXB2 modify contractions of isolated rat cardiac muscle



cells and increased the amount of 45Ca2+ exchanged bynon-beating cells [169]. These results led to the suggestionthat PGI2 could be an endogenous anti-arrhythmic mole-cule [170]. It was reported that PUFAs increased the elec-trical threshold for the induction of ventricular fibrillationthat could reduce the risk of developing malignant cardiacarrhythmias. Mitochondrial dysfunction induced by EFAdeficiency could be eliminated by the presence of normallevels of the essential fatty acids in the ω-3-enriched mito-chondrial membrane phospholipids [171] that mayaccount for the ability of EPA/DHA to decrease cardiacarrhythmias during myocardial ischemia. The recovery ofmitochondrial energy metabolism and myocardial pumpfunction during reperfusion is significantly better in ω-3PUFA-enriched hearts, suggesting that EPA and DHA limitmyocardial injury during ischemia and reperfusion [172].

EPA and DHA (at 2–10 microM) reduced the contractionrate of spontaneously beating, isolated, neonatal rat car-diac myocytes [173] without a significant change in theamplitude of the contractions. Both CO- and LO-inhibi-tors and antioxidants did not alter the effect of the fattyacids. The inhibitory effect of EPA and DHA on the con-traction rate was similar to that produced by the class Iantiarrhythmic drugs. It was also reported that lysophos-phatidylcholine (LPC)- or acylcarnitine-induced arrhyth-mias were completely blocked by EPA, DHA, ALA, AA,and LA by inhibiting the electrical automaticity/excitabil-ity of the cardiac myocytes. Studies using whole-cellpatch-clamp technique in cultured neonatal rat ventricu-lar myocytes revealed that EPA, DHA, and to a limitedextent AA can produce a concentration dependent sup-pression of ventricular, voltage-activated Na+ currents thatmay explain their anti-arrhythmic actions in vitro and invivo [174-176].

PUFAs modulate telomere and telomerase activityTelomere, the genetic segment that appears at the end ofthe chromosomes, has the special property of protectingthese ends. Telomerase is the enzyme that adds telomererepeats to the ends of the chromosomes with the use of adedicated RNA template. Inactivation of telomerase leadsto telomere shortening and eventual senescence of thecells. Telomerase consists of two principal subunits: tel-omerase reverse transcriptase (TERT), the protein catalyticsubunit, and the telomerase RNA component (TERC). Pri-mary cells when grown in vitro, lack sufficient TERT tomaintain telomeres and hence, telomeres shorten progres-sively with each cell division. This eventually results inshorter telomere that loses its ability to protect the ends ofchromosomes and is therefore recognized by the cell'sDNA repair machinery as damaged DNA. The loss of tel-omere results in cellular senescence since cell can nolonger divide and replicate itself. In contrast, overexpres-sion of TERT prevents telomere attrition and enables cells

to proliferate indefinitely, a character of cancer cells. Thus,telomere and telomerase are central to several diseasessuch as cancer, aging, atherosclerosis, CHD, type 2 diabe-tes, hypertension, and to the biology of stem cells.

Recent reports suggested that leukocyte telomere length isa predictor of future CHD in middle-aged, high-risk men,whereas 10 mg of pravastatin, a HMG-CoA reductaseinhibitor, substantially abrogated shortening of the tel-omere length in high-risk subjects [177-179], suggestingthat patients with CHD have senescent endothelial cells.Telomere shortening has been reported in patients withtype 2 diabetes mellitus, hypertension, and insulin resist-ance [180-184]. Diabetes mellitus, hypertension, insulinresistance, and CHD are not only low-grade systemicinflammatory conditions in which plasma levels of lipidperoxides, IL-6, and TNF-α are increased and eNO andconcentrations of anti-oxidants are decreased, but alsoshow shorter leukocyte telomere length compared to con-trols. NO activates telomerase [185] and delays endothe-lial cell senescence [186], whereas asymmetrical dimethylarginine, an inhibitor of NO synthesis, enhances endothe-lial cell senescence [187,188]. It was reported that stableexpression of hTERT (human telomerase reverse tran-scriptase) enhances production of eNO and NO activityand renders endothelial cells to show younger phenotype[189,190], whereas NO activates telomerase and delaysendothelial cell senescence. These results imply that NOprevents whereas reactive oxygen species induce telomereshortening.

In contrast, tumor cells express increased telomerase activ-ity. Tumor cells have relatively higher content of anti-oxi-dants and reduced concentrations of lipid peroxides dueto PUFA deficiency. It is known that ω-3 PUFAs are of ben-efit in type 2 diabetes, hypertension, and hypertriglyceri-demia and prevent CHD, in part, by enhancing NOgeneration from endothelial cells and decreasing insulinresistance [191]. Tumor cells undergo apoptosis on expo-sure to ω-3 PUFAs (especially in response to EPA, DHA,and GLA) due to increase in intracellular free radical gen-eration and formation of lipid peroxides. Since, NO andlipid peroxides modify telomerase activity, it is likely thatPUFAs enhance or decrease activity of TERT in endothelialcells and tumor cells respectively. This is supported by theobservation that EPA and DHA inhibit hTERT activity inhuman colorectal adenocarcinoma cells [192,193]. Thus,PUFAs can prevent, reverse or arrest atherosclerosis andCHD by their ability to enhance eNO synthesis that, inturn, augments hTERT activity and prevents endothelialsenescence.

ConclusionIt is evident from the preceding discussion that PUFAs,especially an optimal combination of EPA, DHA and pos-



sibly, GLA, DGLA and AA show most the qualities of thesuggested "polypill", viz., aspirin-like action, inhibition ofHMG-CoA and ACE enzymes, and possess diuretic, anti-hypertensive, and β-blocker-like actions (see Table 1 for asummary of actions). PUFAs are naturally occurringendogenous substances, present in almost all tissues andare essential components of all mammalian cells and havebeen shown to be relatively safe when administered to dif-ferent types of patients for long periods of time (from fewmonths to few years). This is evident from the fact thatEskimos consume large amounts of marine fish that arerich in ω-3 fatty EPA and DHA and are not known to sufferfrom any significant side effects. Nevertheless, possibleside effects due to long-term feeding of PUFAs need to bestudied. One concern that is generally expressed aboutPUFAs is that their increased intake may enhance lipidperoxidation, and that these oxidized products could beharmful to tissues. But, it was reported that increasedintake of EPA/DHA, in fact, reduces in vivo lipid peroxida-tion and oxidative stress in humans [194-196]. Further-more, peripheral leukocytes, which are major mediatorsof inflammation, are capable of de novo production of cat-echolamines that enhance the inflammatory response[197]; whereas vagal parasympathetic signaling sup-presses inflammation through cholinergic receptors onthese cells [138]. This suggests that sympathetic and para-sympathetic pathways and immune system cross talk witheach other during inflammation. PUFAs, especially ω-3fatty acids enhance acetylcholine levels [135-137] andincrease HRV [133,134] due to their ability to augmentparasympathetic tone and thus, indirectly function asendogenous suppressors of sympathetic nervous systemand thus, of β-receptor function. This is especially so sinceunder normal conditions a balance is maintainedbetween sympathetic and parasympathetic systems andpro- and anti-inflammatory pathways. These evidencesimply that PUFAs function as endogenous enhancers ofparasympathetic tone, suppress inflammatory events; andinhibit sympathetic over activity, and block β-receptoraction.

In view of these beneficial actions (see Table 2 for a sum-mary of their actions), ω-3 and ω-6 fatty acids can be givenfor the prevention of CVD. Since PUFAs can be given topregnant women and lactating mothers, and children, it issuggested that a combined ω-6 and ω-3 pill could be givenfrom childhood. Furthermore, several studies suggestedthat PUFAs, especially ω-3 fatty acids, are useful in the pre-vention and treatment of Alzheimer' disease, schizophre-nia, and depression [198-209], suggesting that PUFAshave a much wider benefit compared to the "polypill". Itmay also be mentioned here that for their physiological/beneficial action(s) PUFAs need many co-factors such asfolic acid, vitamin B12, vitamin B6, vitamin C, tetrahydro-biopterin (H4B), zinc, magnesium, calcium, L-arginine,and small amounts of selenium and vitamin E [18].Hence, it is essential that these co-factors should also beprovided in adequate amounts to bring about the benefi-cial action of ω-6 and ω-3 PUFAs. Since statins, glitazones,several anti-hypertensive and anti-arrhythmic drugs seemto be mediate their actions by modulating EFA/PUFAmetabolism, it is possible that sub-clinical deficiency oraltered metabolism of EFAs may subvert their actions/benefits. Hence, it is prudent to provide a combination ofω-6 and ω-3 PUFAs and their co-factors along with statins,glitazones, and other drugs in the treatment of CVD.Yokoyama et al [166] showed that long-term use of ethylEPA (1800 mg/day) produced a significant reduction innon-fatal coronary events in patients with dyslipidemiacompared to control. This risk reduction occurred after 2.5years of use of ethyl EPA (mean follow up period was 4.6years) even when PUFAs were added in addition to statintreatment lending support to the concept that ω-3 and ω-6 PUFAs can be combined with other cardiovascular drugsin the prevention and treatment of cardiovascular dis-eases.

Competing interests UND owns and runs the biotech company UND Life Sci-ences that specialises in developing lipid-based drugs forcancer, diabetes melltus and hypertension.

Table 1: Possible cumulative impact of four secondary prevention treatments in the prevention of cardiovascular diseases.

Drug therapy Relative-risk reduction 2-year event ratio

None - 8%Aspirin 25% 6%B-blockers 25% 4.5%Lipid lowering (by 1–5 mmol) 30% 3.0%ACE inhibitors 25% 2.3%

Cumulative relative reduction if all four drugs are used is about 75% [see ref. [91]].Events that were included in this analysis are: cardiovascular death, myocardial infarction or strokes.



Author's Contributions I contributed to everything.

AcknowledgementsUND was in receipt of Ramalingaswami Fellowship of Department of Bio-technology, New Delhi, India during the tenure of this study.

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Table 2: Actions of PUFAs (especially of ω-3 fatty acids) that account for their beneficial actions in inflammation, atherosclerosis, hypertension, hyperlipidemias, type 2 diabetes mellitus and coronary heart disease.

Target Effect

Plasma triglyceride concentration-fasting and post-prandial ↓↓Plasma cholesterol ↓↔HDL cholesterol ↑↔LDL cholesterol ↓↔Blood pressure ↓Diuretic-like action ↑Endothelial production of NO ↑ACE activity ↓HMG-CoA activity ↓Platelet aggregation ↓Leukocyte activation ↓Cardiac arrhythmias ↓Heart rate variability ↑Production of lipoxins and resolvins ↑Formation of lipid peroxides ↓Production of PGI2, PGI3, PGE1 ↑Production of TXA2, LTs ↓Synthesis of pro-inflammatory cytokines such as TNF-α and MIF ↓Production of anti-inflammatory cytokines such as IL-10 ↑Insulin sensitivity ↑Endothelial integrity ↑Telomere length ↑Parasympathetic tone ↑Sympathetic tone ↓

































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